Innature,bacterialiveincommunitiesthatinvolvecomplexintercellularinteractionsthatgovern higher-orderprocessessuchasbiofilmformation,virulenceandevolution.Communicationbetween bacteriaisimportantforthedevelopment,architectureandsurvivalofthesecommunities. Communicationisgenerallymediatedbysecretedproteins,ligandsandtheirsurfacereceptors,which transmitthesignalsbetween,andinto,cells.Nothingisknownofintercellularsignalingin mycobacteria,despitethelikelyimportanceofcommunicationincoordinatingcommunalactivitiesin theenvironment,inbiofilmsandininfection.Ourgoalinthisproposalistoaddressthisgapinour knowledgeand,thereby,begintounderstandhowandwhymycobacteriacommunicate. ConjugaltransferofchromosomalDNAoccursfromdonortorecipientstrainsofMycobacterium smegmatisbyadistinctiveprocesscalleddistributiveconjugaltransfer(DCT).UtilizingRNA-seq,we recentlydescribedtranscriptionalresponsesindonorandrecipientcellsthatwerecontactdependent. Notably,thiscontact-dependentsignalingresultedintheinductionandrepressionofmanygenes (>100)inbothcelltypes,manyofwhicharenotassociatedwithDNAtransfer.Giventheubiquitous natureofcell-cellcommunicationinbacteria,wehypothesizethatthesenon-DCTtranscriptional responsesreflectotherimportantformsofintercellularsignalingandthatthiscommunicationwillbe prevalentinco-culturesofothermycobacterialspeciesandbetweenspecies.Wewilltestthis hypothesisbydeterminingtheco-culturedependent,transcriptionalresponsesoffast-andslow- growingmycobacteria,includingM.abscessusandM.canettii(Aim2). ThesignalinginM.smegmatisrequiredtheTypeVIIsecretionsystem,ESX-1,intherecipientstrain andresultedininductionoftherelatedesx4secretionlocus.Notably,esx4wasinducedonlyinthe recipientanditslevelofinductionwasresponsiveintranstotheESX-1activityofthedonor.These studieswerethefirsttoidentifyabiologicalroleforESX-4,andimportantly,showedthatitisonly expressedinresponsetocell-cellcontact.Ourpreliminarydatashowthattheextra-cytoplasmic sigmafactor,SigM,isresponsiblefortriggeringesx4expression.Moreover,theSigMregulon includesgenesnearesx4thatarealsorequiredforDCT,indicatingthatafunctionalesx4locus extendsbeyondtherecognizedcorelocus.Wewilldefinethecontact-dependentSigMregulonthat drivesesx4expression,whichisrequiredforDCT(Aim1).Thisanalysiswillalsoservetoillustrate thepowerofourapproachinfirstidentifyingandthencharacterizingothercoculture-dependent responsesinmycobacterialcommunities.